Antifreeze polypeptide-expressing microorganisms useful in fermentation and frozen storage of foods

ABSTRACT

Methods and compositions for preparation of frozen fermented food products using antifreeze polypeptide-expressing microorganisms are provided. In particular the invention provides for use of fish antifreeze polypeptide-expressing microorganisms in fermentation of milk to produce and store frozen yogurt.

This is a continuation of application Ser. No. 08/321,991, filed Oct.12, 1994, now U.S. Pat. No. 5,676,985 the disclosure of which isincorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to methods and reagents usefulin maintaining the quality of frozen food products during frozenstorage, particularly enhanced storage life and the maintenance ofconsumer accepted quality of dairy products.

BACKGROUND OF THE INVENTION

Refrigeration, particularly freezing, is a common and preferred meansfor storing biological materials. Frozen storage generally arrests orconsiderably slows the deterioration of the biological product.

Frozen or refrigerated foods are now a mainstay of the human diet indeveloped nations. Thus extensive research has and is being carried outby food scientists to ensure high quality products for the consumers.This is particularly true with regard to frozen vegetables and frozendeserts such as ice cream and yogurt.

Frozen deserts such as ice cream or yogurt are generally eaten in thefrozen state. Thus, the texture of the frozen product as well as itsflavor is important to consumers. Texture is to a large extent governedby the size of the ice crystals. Producers of these frozen deserts havegone to considerable effort and expense to ensure smooth texturedproducts. However, during frozen storage the ice crystals can grow andthus roughen and spoil this texture. The growth of ice crystals duringfrozen storage is known as recrystallization. This problem isparticularly common when the frozen storage conditions are less thanideal, such as during transportation or storage in modern frost-freehome freezers. After a relatively short period of time at above-zerotemperatures (i.e., above 0° C.), or even at sustained freezingtemperatures, frozen foods can become less desirable or even unsuitablefor human consumption due to the ice recrystallization process.

Although manufacturers use a variety of techniques to reduce the damageassociated with recrystallization success has been limited andsignificant problems remain. Thus there is a need for new techniques toreduce or prevent the recrystallization process and improve thecharacteristics of frozen foods. These techniques and compositionsshould be inexpensive and completely safe and suitable for humanconsumption.

It has been clearly demonstrated that antifreeze polypeptides (AFP) caneffectively inhibit ice recrystallization at low (μg/ml) concentrationsin aqueous solutions and frozen food products (see, e.g., Knight et al.,1984, Nature 308:295-296; Knight et al., 1986, Cryobiology, 23:256-262;Knight et al., 1988, Cryobiology, 25:55-60; Warren et al., U.S. Pat. No.5,118,792). Warren et al, supra, have suggested adding purifiedantifreeze polypeptides directly to food products prior to freezing toimprove preservation characteristics during frozen storage.

At the present time antifreeze proteins are available for commercial usefrom two sources; the blood serum from a small number of fish speciesfound in cold water, and recombinant DNA techniques such as thosedescribed by (but not restricted to) Warren et al., supra. Other sourcesof antifreeze proteins, such as transgenic plants and animals, arecurrently being explored. Regardless of the source, the antifreezepolypeptides must be isolated from the medium in which they are found orproduced, and subject to extensive purification. These purificationprocedures are expensive to the point where the cost of the antifreezepolypeptide additions could exceed the value of the frozen product.Moreover, the purification protocol may introduce contaminants unsafefor consumption.

In the view of the inherent value to producers and consumers ofinhibiting ice recrystallization in frozen fermented dairy products, andthe fact that antifreeze polypeptides are very effective in this regard,it is desirable to develop methods to incorporate the antifreezepolypeptides into the food products in the most efficient and costeffective method possible.

An ideal method of incorporating antifreeze polypeptides into frozenfermented food products is to have the organism responsible for thefermentation process produce the antifreeze proteins while fermentingthe food. A number of antifreeze polypeptides and their genes have beenwell characterized and sequenced (see, e.g. Anathanarayanan, 1989, LifeChem. Rep. 7:1-32 and Davies et al., 1990, FASEB Journal 4:2460-2467).Several of these genes have been expressed in bacteria, transgenicplants, fish and Drosophila (see, e.g., Fletcher et al., 1988, Can. J.Fish. Aquat. Sci. 45:352-357; Rancourt et al., 1987, Mol. Cell. Biol.7:2188-2195; Kenward et al., 1993, Plant Mol.Biol. 23:377-385; Li etal., 1991, Protein Engineering 4:995-1012; and Sönnichsen et al., 1993,Science 259:1154-1157).

In view of the widespread popularity of frozen fermented dairy productssuch as yogurt it would be desirable to develop methods for producingsuch products more efficiently at lower cost and with better flavor andtexture. The present invention provides methods for achieving this goal.

SUMMARY OF THE INVENTION

The present invention provides methods for preparing a frozen fermentedfood product. This method comprises the steps of (a) contacting a foodproduct with a microorganism that is capable of secreting a fishantifreeze polypeptide, wherein the microorganism is capable offermenting the food product to produce the fermented food product, (b)incubating the food product with the microorganism under conditions inwhich fermentation takes place so that a fermented food product isproduced having the antifreeze polypeptide present in an amounteffective at inhibiting recrystallization of the product; and (c)freezing the fermented food product at a temperature below −5° C., so asto produce a frozen fermented food product.

In one embodiment the food product is a dairy product (e.g., milk) whichcan be fermented to produce yogurt, buttermilk or cheese.

The microorganism of the invention is usually a bacterium (e.g.,Lactobacillus bulgaricus; Streptococcus cremoris, Streptococcus lactis;Bifidobacterium bifidum, Bifidobacterium longum) but may also be afungus such as a yeast (e.g., Saccharomyces fragilis, Saccharomycescerevisiae, Saccharomyces lactis, and others). According to theinvention these microorganisms are genetically engineered so that theyare capable of secreting a fish antifreeze polypeptide (or a peptidewith substantial sequence similarity to a fish antifreeze polypeptideand with antifreeze properties similar to a fish antifreezepolypeptide). In a preferred embodiment, the antifreeze polypeptide isan animal antifreeze polypeptide, with a fish antifreeze polypeptidepreferred. Most preferred is microorganism capable of expressing anocean pout type III antifreeze polypeptide (see, e.g., Hew et al., 1984,J. Comp. Physiol. B155:81-85; Li et al., 1985, J. Biol. Chem.260:12904-12909).

In a most preferred embodiment the invention comprises incubating milkwith bacterial species Lactobacillus balgaricus and Streptococcus lactisthat are capable of fermenting milk to produce yogurt and capable ofsecreting an ocean pout type III antifreeze polypeptide; incubating thebacteria and milk under conditions that produce yogurt; and freezing theyogurt at a temperature below −5° C., so as to produce frozen yogurt.

The invention also provides a composition comprising yogurt and amicroorganism wherein the microorganism comprises a gene encoding a fishantifreeze polypeptide.

DETAILED DESCRIPTION Definitions

As used herein, “fermentation” refers to the chemical conversion ofcarbohydrates or proteins in foods through the use of microorganisms. Inthis process carbohydrates are often converted to lactic acid.

As used herein, “food product” refers to a foodstuff (a substance thatcan be used, or prepared for use, as food) that can be transformed bythe action of a fermenting microorganism to a fermented food product.

As used herein “fermented food product” refers to an edible foodprepared by a process that includes fermentation by a microorganism.

As used herein “yogurt” refers to a dairy product produced by the lacticacid fermentation of milk by the action of microorganisms.

As used herein “Antifreeze polypeptides” (AFPs) refers to macromoleculesfound in the body fluids of some animals and plants, which have thecommonly known property that they reduce non-colligatively the freezingpoint of water. Antifreeze polypeptides are also known as “thermalhysteresis proteins.” As used herein, “antifreeze polypeptides” includesglycoproteins as well as chemically synthesized, and recombinantlyproduced polypeptides having a protein sequence with substantialsimilarity to a naturally occurring APP and retaining the properties ofan antifreeze polypeptide.

As used herein “fish antifreeze polypeptide” refers to an AFP that isfound in nature in a fish, as well as chemically synthesized andrecombinantly produced polypeptides having a protein sequence withsubstantial similarity to a naturally occurring fish AFP and retainingthe properties of a antifreeze polypeptides.

As used herein, “recombinantly produced polypeptides” refers to apolypeptide produced using recombinant DNA techniques. Recombinant DNAtechniques are well known and are characterized by the joining of atleast two segments of DNA that are not naturally joined in nature (e.g.,a bacterial promoter and a fish polypeptide coding sequence).

As used herein, “substantial similarity” denotes a characteristic of apolypeptide sequence or nucleic acid sequence, wherein the polypeptidesequence has at least 70 percent sequence identity, preferably 80percent sequence identity, and most preferably 90% sequence identitycompared to a reference sequence (e.g., a naturally occurring antifreezepolypeptide), and the nucleic acid sequence has at least 80 percentsequence identity and preferably 90% sequence identity compared to areference sequence. The reference sequence may be shorter than thefull-length naturally occurring polypeptide or nucleic acid sequence butwill be at least 12 residues long for the case of a polypeptide and atleast 36 bases long for the case of a nucleic acid.

Description

The present invention provides methods for preparing a frozen fermentedfood product by adding a microorganism that is capable of fermenting thefood product to produce the fermented food product and also is able tosecrete a fish antifreeze polypeptide. The use of a microorganism thatboth secretes an AFP and ferments the food product has severaladvantages over other methods for affecting ice crystal formation andfreezing temperature. For example, the claimed method avoids the costlynecessity for purifying an AFP prior to addition to a food product. Inaddition, this will eliminate any possible contamination from thepurification protocol and the pyrogenicity associated with foreignmicroorganisms. Furthermore, because the AFP is secreted by thefermenting microorganism of the claimed invention, this process requiresfewer steps than other methods.

The food product of the invention is usually milk but other foods thatare fermented to produce an edible fermented food may also be used.Examples include cabbage (which can be fermented to produce sauerlraut),cucumbers (which can be fermented to produce pickles) and soybeans(which can be fermented to produce miso and other products).

In one step of the claimed method, the food product is contacted ormixed with a microorganism capable of fermenting the food product.Examples of microorganisms useful in food fermentation are well known(see, e.g., van de Guchte, 1992, FEMS Microbiology Reviews, 88:73-92).

In a preferred embodiment the food product is milk (e.g., from a cow[i.e. bovine], ewe, mare, or goat). The action of fermentingmicroorganisms, typically bacteria, on the milk produces yogurt,buttermilk, or certain cheeses, according to the choice of the bacteriaand the conditions of incubation. In a most preferred embodiment themethod of the invention will be used to produce yogurt from milk. Yogurtis referred to by a variety of names around the world. Table 1 providesa list of the names and country of origin of the common varieties.

Methods for yogurt production can be found in Functions of FermentedMilk edited by Nakazawa and Hosono, 1992, published by Elsevier AppliedScience, London-New York, p. 32, which is incorporated herein byreference. In the United States yogurt is produced from either whole orskim milk from cows. The milk is standardized to 10.5 to 11.5% solids,heated to above 90° C. (30 to 60 minutes) to destroy any contaminatingmicroorganisms, and then cooled. The material is then inoculated with amixed culture of Streptococcus thermophilus and Lactobacillus bulgaricusin a 1:1 ratio. The combined action of these two organisms is usuallyneeded to obtain the desired flavor and acid in the products. In otherinstances, other high fermenting bacteria including bulgarian bacteria,L. jugarti, L. acidophilus, Bifido bacterium, spp. Yeast and lacticfungi have also been used. Examples of bacteria and other organisms usedfor the fermentation of milk to produce yogurt are given in Table 2.

According to the invention, the microorganisms will be geneticallyengineered (i.e., employing the techniques of recombinant DNAtechnology) so that they are able to secrete a fish antifreezepolypeptide. Examples of fish antifreeze polypeptides include type I AFPof winter flounder; type II AFP of sea raven, herring and smelt; typeIII AFP of ocean pout and wolffish, and AFGP (antifreeze glycoprotein)of cods and nototheniids (see, e.g. Davies et al., 1990 FASEB Journal4:2460-2467, and Ewart et al., 1992, Biochem. Biophys. Res. Comm.185:335-340).

Most preferred is microorganism capable of expressing an ocean pout typeIII antifreeze polypeptide. The ocean pout type III antifreezepolypeptide is preferred because it has no amino acid bias and has beenshown to be active when expressed in E. coli (Li et al., 1991, ProteinEngineering 4:995-1012; Sönnichsen et al., 1993, Science,259:1154-1157). In addition, the type III AFP is preferred because typeI AFP of winter flounder may not be stable at the fermentationtemperature and type II AFP may not be correctly folded in bacterialsystem and is very susceptible to reduction.

The methods for engineering bacteria and fungi capable of expressing andsecreting a heterologous polypeptide are well established (see, e.g.,Maniatis et al. (1982), Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, New York; Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology 152(Academic Press, Inc., San Diego, Calif.); Simon et al., 1986, Appl.Environ. Microbiol. 52:394-395; and von Wright et al., 1985, Appl.Environ. Microbiol. 50:1100-1102, all of which are incorporated hereinby reference)

The production of microorganisms capable of expressing and secreting anAFP can be carried out in a variety of ways that will be apparent to oneof ordinary skill. The DNA sequence encoding the AFP will preferably beoperably linked (i.e., positioned to ensure the functioning of) to anoperon which allows the DNA to be transcribed (into an RNA transcript)and translated into a polypeptide in the microorganism. Promoters forboth bacteria and fungi are well known in the art. Preferred operons forexpression in lactic acid bacteria include the lactose operon of S.thermophilus or lac ABCDFEGX operon of L. lactic because they have beenused successfully to drive foreign gene expression in the hosts (see,e.g., Simons et al., 1993, J. Bact. 175:5186-5175; Mollet et al., 1993,J. Bact. 175:4315-4324).

The AFP may be expressed as a fusion polypeptide for increased stabilityor other beneficial properties. Furthermore the AFP polypeptide may bemodified via a modification of the gene encoding the polypeptide. Ingeneral, modifications of the genes may be readily accomplished by avariety of well-known techniques, such as site-directed mutagenesis(see, e.g., Gillman and Smith, 1979, Gene 8:81-97 and Roberts et al.,1987, Nature 328:731-734).

The microorganisms of the invention are capable of secreting the AFP.Accordingly, the AFP will preferably be linked to a signal peptidesequence. Examples of suitable signal peptide sequences include thosefrom the usp45 gene of L. lactis ssp lactis MG 1363 and the L. lactisssp cremoris SK 11 cell envelop associated protease gene (van Asseldonket al., 1990, Gene 95:155-160; De vos et al., 1989, J. Dairy Sci.72:3398-3405). For bacteria such as L. lactis the usp45 signal peptideis preferred since it derives from the same host. In one preferredembodiment the AFP gene is linked to a transcription terminationsequence to ensure correct termination of AFP transcription in the hostsystem.

An AFP gene construct including elements described above is constructedusing plasmids such as pUC19, pNZ18 and pDBN183 as vectors (Solaiman etal., 1992, Plasmid, 28:25-36). The AFP gene construct is incorporatedinto the genome of a lactic acid bacterial species using homologousrecombination techniques (Mollet et al., 1993, J. Bact., 175:4315-4324).The lactic acid bacteria and E. coli strains can be maintained asrecommended by Maniatis et al. in Molecular Cloning, A LaboratoryManual, supra; and Chagnand et al., 1992, Can. J. Microbiol. 38:67-74.

Fish antifreeze-expressing (FAE) microorganisms may be applied to foodproducts in any conventional way. In the case of products such as milk,the bacteria or fungus can be mixed intimately with the foodstuff thatis to be fermented and frozen. It will be known by those of skill thatmixtures of different microorganisms are sometimes used to produce thedesired product. For example, in preparation of yogurt, S. thermophilusand L. bulgaricus are often used together.

The number of FAE microorganisms added to the food product will dependon the properties of the microorganisms and of the food. Generally,lactic acid FAE starter bacteria (10¹⁰-10¹¹ per ml) are incubated at1-5% into pasteurized and cooled milk such that the proportion resultsin an appropriate amount of antifreeze polypeptide in the product. Theamount of AFP in the product should be an amount effective at preventingor inhibiting ice recrystallization (1-100 mg/liter milk). This can bedetermined using the splat-cooling assay described by Knight et al.(1988) Cryobiology, vol. 25, pp. 55-60.

In another step of the method, the fermented food product is frozenusing conventional freezer operations, such as blast freezers (−20° to40° C.) or contact plate freezers (−30° to 40° C.) or vacuum freezedriers. It will be apparent to one of ordinary skill that numerousvariations of the aforementioned embodiments are possible.

TABLE 1 Names used to describe types of yogurt Product Name Country ofOrigin Jugurt/Eyran/Ayran Turkey, etc. Busa Turkestan Kissel MlekaBalkans Urgotnic Balkan Mountains Leban/Laban Lebanon/Arab countriesZabady (Zabbady) Egypt/Sudan Mast/Dough Iran/Afghanistan Roba IraqDahi/Dadhi/Dahee India Mazun/Matzoon/Matsun/ Armenia Matsoni KatykTranscaucasia Tiaourti Greece Cieddu Italy Mezzoradu Sicily GiodduSardinia Biokys Czechoslovakia Karmdinka Poland Tarho HungaryTykmaelk/Ymer Hungary Villi (Fiili) Finland Filmjolk/Fillbunke/Scandinavia Surmelk/Taettemjolk/ Tettemelk logurte Brazil/PortugalProghurt Chile Skyr Iceland Gruzovina Yugoslavia Kefir/Donskaya/VarentesSoviet Union Kurunga/Koumiss/ Ryazhenka/Guslyanka Tarag MongoliaShosim/Sho/Thara Nepal

TABLE 2 Microorganisms commonly used in fermented milk and lactic drinksGenus Habit Fermentation Main Species Streptococcus^(a) Coccal chainsHomo S. cremoris, lacfis, thermophilus Leuconostoc^(b) Coccal pairsHetero L. citrovorum, mesenteroides Lactobacillus^(c) Rods Homo L.acidophilus, bulgaricus, casei, jugurti, lactis Bifidobacterium RodsHetero B. bifidum, breve, longum Others: Yeasts (Torulopsis holmil;Saccharomyces fragilis, cerevisiae, lactis; Candida pseudotropicalis,etc.) Fungi (Geotrichum candidum) Acefic acid bacteria (Acetobacteracefi, rasens) ^(a)Now Lactococcus lactis subsp. cremoris, Lac, lactissubsp. lactis and S. thermophilus. ^(b)Now L. mesenteroides subsp.cremoris and L. mesenteroides subsp. mesenteroides. ^(c)Now L.acidophilus, L. delbrueckii subsp. bulgaricus, L. casei subsp. casei, L.helveticus biovar. jugurti and L. delbrueckii subsp. lactis.

EXAMPLES

The invention is illustrated by the following examples. These examplesare offered by way of illustration, not by way of limitation.

Example 1

Construction of a strain of lactic acid bacteria that produce antifreezepolypeptide—Method I

To engineer a lactic acid bacterium that produce antifreeze proteinsseveral steps are involved. The first step is selection and preparationof a chromosomal site for the AFP gene integration. A native operon of astrain of lactic acid bacteria such as the lactose operon of the S.thermophilus or L. lactis genome, consisting of the lacS (lactosepermease) and lacZ (β-galactosidase) genes, is used for the integrationof an antifreeze protein gene. Integration of an AFP gene into such anoperon should preserve its correct function. The AFP gene should becomea functional part of the operon and be regulated similarly (see, e.g.,Simons et al., 1993, J. Bact., 175:5186-5175, and Mollet et al., 1993,J. Bact. 175:4315-4324).

To do this, the lacS and lacZ genes from the host bacteria are cloned byPCR procedures or conventional gene cloning methods. At least onerestriction enzyme site is generated between the two genes by designingparticular primer sequences for the PCR reactions. The restrictionenzyme sites are generated for convenient segment linkage and insertionof DNA fragment. An antibiotic resistance marker gene such as anampicillin or erythromycin resistance gene is inserted into thegenerated restriction enzyme site. The lacS-Amp™-lacZ DNA in anappropriate vector such as pNZ932 is transformed into the lactic acidbacterial strain (see, e.g. Simons et al., 1993, J. Bact.175:5186-5175). Ampicillin-resistant transformants will be selected, andgene integration will be verified using PCR and DNA sequencing.

The second step involves construction of an antifreeze protein genecassette. Using the nucleotide sequence derived from a cloned ocean pouttype III AFF gene, an appropriate type III AFP gene is assembled fromsynthetic oligonucleotides using the preferential codons of the host(see, e.g., Mercenier, 1990, FEMS Microbiology Reviews, 87:61-78, andvan Asseldonk et al., 1992, FEMS Microbiology Reviews 88:73-92). To makea bacteria secrete AFP, a signal peptide sequence (SP) from homologousgenes such as the usp45 gene of L. lactis ssp lactis MG 1363 and the L.lactis ssp cremoris SK11 cell envelop-associated protease genes is fusedto the 5′-end of the type III mature AFP coding sequence (van Asseldonket al., 1990, Gene 95:155-160, and De vos et al., 1989, J. Dairy Sci.72:3398-3405). The AFP gene cassette, including a signal peptidesequence and an AFP gene will be inserted between the lacS and lacZgenes in vitro to generate a lacS-SP-AFP-lacZ construct.

Finally, the lacS-SP-AFP-lacZ DNA is incoporated into the genome of theampicillin-resistant bacteria generated from step one by homologousrecombination techniques. Gene replacement of lacS-Amp™-lacZ bylacS-SP-AFP-lacZ is initially selected by their ability to grow onmedium in the presence or absence of ampicillin. AFP gene integration isconfirmed by inverse PCR, Southern blot analysis and DNA sequencing.

Example 2

Construction of a strain of lactic acid bacteria that producesantifreeze polypeptide—Method II

An alternative method to generate an antifreeze protein-producing lacticacid bacterium is to eliminate the step of integrating an antibioticresistance gene construct into the genome of the bacterium. In thiscase, experiments will be carried out as described in steps 2 and 3 inExample 1. A labelled AFP gene fragment will be used to identifyrecombinant clones, and AFP gene integration will be further confirmedby inverse PCR and DNA sequencing.

Example 3

Use of antifreeze polypeptide expressing bacteria in yogurt production

The genetically engineered bacterium can be directly used to preparestarters for making frozen yogurt. In brief, a small quantity of thegenetically engineered stock culture (frozen or cold stored culture) iscultured in 0.5-1 liter of pasteurized skim milk to prepare a motherculture. Intermediate fermentation is then carried out to scale up10-100 times by stages from the mother culture to the bulk starter whichcan be added directly to pretreated milk. Alteratively, bulk starter canbe prepare directly from frozen stocks. The milk mixture is prewarmed to55-60° C. and then homogenized (150-200 Kg/cm²) and pasteurized (90-95°C. for 5-10 minutes or 120-130° C. for a few seconds), and cooled to atemperature range within which the lactic acid bacteria will not bedamaged (45-48° C.). The starter bacteria are applied to inoculate withpretreated homogenized milk at 30-45° C. When the acidity reaches acertain prescribed level such as 0.8% and the amount of AFP in theproduct reaches required concentration (1-100 mg/liter milk), thefermented milk is cooled to 15-20° C. to suppress bacterial activity.These products are used to make soft frozen yogurt, hard frozen yogurt,and mousse yogurt by adding different percentages of fruit syrup, sugar,stabilizers, fruit juice and emulsifiers to a cold fermented milk base.

Example 4

Production of frozen yogurt

Soft frozen yogurt is made by adding 20% fruit syrup and stabilizers andemulsifiers to 80% of a cold fermented milk base, and then filling intocontainers with a. 50-60% overrun using a normal ice cream freezer. Theproduct is stored at 0-6° C.

Hard frozen yogurt is made of 35% fruit juice, the overrun is 70-80%,and storage temperature is below −25° C. Mousse yogurt is made by mixinga fermented milk base with a warm mousse base (a homogenized mixture ofskim milk, sugar, stabilizers and emulsifiers). The product is stored atbelow 0° C.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

What is claimed is:
 1. A method for preparing a frozen fermented foodproduct comprising the steps of: a) contacting a food product with amicroorganism that secretes an antifreeze polypeptide into the foodproduct and that ferments the food product; b) incubating the foodproduct with the microorganism under conditions in which fermentationtakes place so that a fermented food product is produced having theantifreeze polypeptide present in an amount effective at preventing orinhibiting ice-crystal formation in the food product during storage;and, then c) freezing the fermented food product at a temperature below−5° C. so as to produce a frozen fermented food product.
 2. The methodof claim 1 wherein the food product is a dairy product.
 3. The method ofclaim 2 wherein the food product is bovine milk.
 4. The method of claim3 wherein the fermented food product is yogurt.
 5. The method of claim 3wherein the fermented food product is selected from the group consistingof buttermilk and cheese.
 6. The method of claim 1 wherein themicroorganism is a bacterium.
 7. The method of claim 6 wherein thebacterium is selected from the group consisting of Streptococcuscremoris, Streptococcus lactis, Streptococcus thermophilus, Leuconostoccitrovorum, Leuconostoc mesenteroides, Lactobacillus acidophilus,Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus jugurti,Lactobacillus lactis, Bifidobacterium bifidum, Bifidobacterium breve,and Bifidobacterium longum.
 8. The method of claim 1 wherein themicroorganism is a fungus.
 9. The method of claim 8 wherein the fungusis a yeast.
 10. The method of claim 9 wherein the yeast is selected fromthe group comprising Torulopsis holmil, Saccharomycesfragilis,Saccharomyces cerevisiae, Saccharomyces lactis, and Candidapseudotropicalis.
 11. A method for preparing frozen yogurt comprisingthe steps of: (a) contacting milk with a microorganism that secretes anantifreeze polypeptide into the milk and that ferments the milk; (b)incubating the milk with the microorganism under conditions in whichfermentation takes place so that yogurt is produced having theantifreeze polypeptide present in an amount effective at preventing orinhibiting ice-crystal formation in the yogurt during storage; and, then(c) freezing the yogurt at a temperature below −5° C. so as to producefrozen yogurt.
 12. The method of claim 11 wherein the microorganism is amember selected from the group consisting of Streptococcus cremoris,Streptococcus lactis, Streptococcus thermophilus, Leuconostoccitrovorum, Leuconostoc mesenteroides, Lactobacillus acidophilus,Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus jugurti,Lactobacillus lactis, Bifidobacterium bifidum, Bifidobacterium breve,Bifidobacterium longum, Torulopsis holmil, Saccharomyces fragilis,Saccharomyces cerevisiae, Saccharomyces lactis, and Candidapseudotropicalis.
 13. A composition comprising a frozen fermented foodproduct containing a microorganism which ferments said food product andwhich secretes an antifreeze polypeptide into said food product.
 14. Thecomposition of claim 13 wherein the microorganism is a bacteriumselected from the group comprising Streptococcus thermophilus andLactobacillus bulgaricus.
 15. A microorganism host cell transformed withan expression vector comprising a first nucleic acid which encodes asignal peptide sequence operably linked to a second nucleic acidencoding an antifreeze polypeptide.
 16. A microorganism host cell ofclaim 15, wherein said microorganism host cell has a cell membrane andsaid antifreeze polypeptide is secreted outside said cell membrane. 17.A microorganism host cell of claim 15, wherein said antifreezepolypeptide is secreted into a medium external from said microorganismhost cell.
 18. A microorganism host cell of claim 15, wherein saidmicroorganism is a bacterium.
 19. A microorganism host cell of claim 18,wherein said bacterium is selected from the group consisting ofStreptococcus cremoris, Streptococcus lactis, Streptococcusthermophilus, Leuconostoc citrovorum, Leuconostoc mesenteroides,Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacilluscasei, Lactobacillus jugurti, Lactobacillus lactis, Bifidobacteriumbifidum, Bifidobacterium breve, and Bifidobacterium longum.
 20. Amicroorganism host cell of claim 19, wherein the bacterium host cell isselected from the group consisting of Streptococcus thermophilus andLactobacillus bulgaricus.
 21. A microorganism host cell of claim 15,wherein said microorganism is a fungus.
 22. A microorganism host cell ofclaim 21, wherein said fungus is a yeast.
 23. A microorganism of claim22, wherein said yeast is selected from the group consisting Torulopsisholmil, Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyceslactis and Candida pseudotropicalis.
 24. A microorganism host cell ofclaim 15, wherein said antifreeze polypeptide is substantially similarto a fish antifreeze polypeptide.
 25. A microorganism host cell of claim24, wherein said antifreeze polypeptide is an ocean pout type IIIantifreeze polypeptide.
 26. A microorganism host cell of claim 15,wherein said expression vector comprises a lacS-SP-AFP-LacZ geneconstruct.